The long term goals of this work are to study functional roles of retinoic acids (RA) and their receptors (RAR) in regulating fetal lung development by controlling lung specific gene expression in respiratory epithelial cells, and to apply the knowledge to clinical treatment. Surfactant protein B (SP-B) maintains alveolar stability by enhancing the rate of spreading of phospholipid at the air-water interface, and therefore is critical to postnatal respiratory adaptation and recovery of respiratory epithelium following oxidant injury. In this application, we propose to determine molecular mechanisms by which RA and RAR stimulate the SP-B gene both in vitro and in vivo. The specific aims are: 1) Identification of cis-retinoic acid responsive elements (RAREs) on the SP-B promoter. DNA footprint, gel electrophoresis mobility shift assay and site-specific mutagenesis will be performed. The studies will be done in respiratory epithelial cells, including the H441 cell line, the ML3-15 cell line and primary isolates of alveolar Type II epithelial cells; 2) Characterization of the interaction between RAR and the tissue-specific transcription factor, TTF-1. Co-immunoprecipitation, Western blot and the yeast two-hybrid system will be performed to identify the precise interaction domains of both RAR and TTF-1, and their roles in binding and activating cis-acting elements in the SP-B gene; 3) Characterization of cis-RARE to determine the effects of RA/RAR on the temporal and spatial expression of the SP-B gene in transgenic mice. The wild type and RARE site-mutated hSP-B promoters will be linked to the CAT reporter gene and introduced into mice. In situ hybridization of tissue sections and CAT assay from transgenic lungs will be used to assess the in vivo RARE hybridization of tissue sections and CAT assay from transgenic lungs will be used to assess the in vivo RARE functional roles determining SP-B promoter expression levels and specificities in bronchiolar and alveolar Type II epithelial cells.